Ready-to-use multiplex real time PCR assays
for the detection of STEC & the identification of EHEC
Sylvie HALLIER-SOULIER(1), Patrick FACH(2) and Lothar BEUTIN(3)
(1)GeneSystems,
centre d’affaires CICEA, 1, rue du courtil, 35170 Bruz, France;
(2)AFSSA,
23, Av du Général de Gaulle, 94700 Maison-Alfort, France;
INTRODUCTION
OBJECTIVE
Shigatoxin-producing Escherichia coli (STEC) have
clearly emerged as important life-threatening foodborne pathogens due to their implication in several
human outbreaks in various countries worldwide.
The most prevalent E. coli serovars associated with
HUS are O26, O103, O111, O145 and O157H7. These
strains carry virulence-associated genes, encoding
for the shigatoxins (stx1 and/or stx2), the intimin
(eae) and/or the enterohemolysin (ehxA). Specific
primers and probe sets for stx (2), eae (1), ehx
genes and the 5 major serogroups were previously
designated (2, 3).
Two innovative multiplex PCR
assays (GeneDiscs), using a
new real time PCR technology
(the GeneDisc Cycler) have
been developed for the routine
detection of STEC and the
identification
of
EHEC
in
foodstuff. The specificity of the
PCR
reactions
was
demonstrated.
(3)Federal
Institute for risk Assessment (BfR), National Reference Laboratory for E. coli, Diedersdorfer Weg 1, Berlin, Germany.
RESULTS
METHOD
Screening GD
Take a fraction of a
single colony
Transfer into
200 µL of
sterile water
Target gene
Number of
strains(1)
ehx+
ehx-
22
32
22
33
34
16
181
33
31
16
98.9
87
162(3)
81
32
221
32
stx1
stx2
stx1 + stx2
stxeae+
eaerfbEO157+
rfbEO157-
Boiling for 10 min
MATERIAL
Identification GD
GeneDisc
Specificity
positive result
(%)
Number of
strains(1)
GeneDisc
positive result
Specificity
(%)
ihp1-O145+
ihp1-O145-
30
327
28
99.4
fliC-H7+
fliC-H7-
24
252
24
100
wzx-O26+
wzx-O26-
29
332
29
100
97.6
wbdl-O111+
wbdl-O111-
23
333
23
100
100
wzx-O103+
wzx-O103-
28
325
28
100
100
(2)
Target gene
(1)Bacterial
(2)
1 Analysis sector/DNA sample
(6 identical sectors/GeneDisc)
6
1
4
3
5
6 PCR wells/DNA sample
2
Screening GD
6
1
4
3
+
5
E. coli O157 & STEC
Identification GD
2
Genotypes were defined by the presence of genes (classical PCR) and the gene expression (Vero cell test, ELISA).
E. coli strains and 30 other Enterobacteriaceae (Yersinia, Salmonella, Citrobacter, Hafnia, Klebsiella, Proteus, Shigella …).(3)E. coli strains and 32 other Enterobacteriaceae.
• The major ehx-types associated with highly virulent EHEC
stains were all detected.
• All genetic variants of stx1 & stx2 were detected except
stx2f (3 strains) which is mainly associated with STEC from
feral pigeons.
EHEC Identfication
• The major eae-types associated with highly virulent EHEC
stains were all detected, except strains carrying the eae-rho
variant (6 strains).
E. Coli O157 & STEC GeneDisc
EHEC Identification GeneDisc
• All types of E. coli O157 , including O-rough mutants (4
strains), were detected by the rfbE gene PCR.
PCR Well
FAM Detection
ROX Detection
PCR Well
FAM Detection
ROX Detection
1
Positive control
Negative control
1
ihp1-O145
Negative Control
2
ehx
stx1 & stx2
2
ihp1-O145
Positive control
3
ehx
stx1 & stx2
3
fliC-H7
wbdl-O111
4
rfbEO157
eae
4
flic-H7
wbdl-O111
5
rfbEO157
eae
5
wzx-O26
wzx-O103
6
Negative control
Positive control
6
wzx-O26
wzx-O103
Screening GD
• The O145 ihp1-like PCR is 100% specific for detection of
EHEC and EHEC-like O145 strains and their O-rough
derivatives.
• The O145 ihp1-like PCR is negative with O145 EPEC, other
non EHEC O145 strains and non-O145 strains except E. coli
O133 and O137, which are stx and eae negative.
• The fliC-H7, wzx-O26, wzx-O103 and wbdl-O111 markers
are 100% specific for E. coli strains, as well as:
- non motile derivatives carrying a fliC-H7 gene,
- O-rough mutants of E. coli O26, O103 and O111.
CONCLUSIONS
Identification GD
This study demonstrates the specificity of the PCR reactions over a large range of bacteria (more than 300
strains). Both GeneDisc assays (screening + identification GeneDiscs) offer reliable tools for the routine
screening of STEC and EHEC in bacterial isolates. Validation of the method is in progress with food samples.
GeneDisc Cycler
REFERENCES
(1)Møller Nielsen E. and Thorup Andersen M., J clin Microbiol, 2003, 41:2884-2893; (2)Perelle S. et al., Mol Cell Probes, 2004, 18:185–192;
(3) Perelle S. et al. J Appl Microbiol, 2005, 98:1162–1168.